BioLife Technologies (BLT) was incorporated on March 31, 1998. BLT's primary mission is to develop improved hypothermic and cryopreservation solutions designed to maintain cells, tissues and organs in a near state of suspended animation. Cardioplegia, organ transplant and selected non- regulated markets have been targeted for development. This Phase I SBIR proposes to use four cardiovascular cell strains as in vitro tools to help develop a future HTS solution that is optimized for human blood vessel preservation. BLT's hypothermic solutions, the HypoThermosol(R) (HTS) series, are better at cold-protecting kidney, heart and skin cells than is ViaSpan(R) - a product produced by DuPont-Merck ten years ago. ViaSpan(R) currently commands the largest market share in the preservation solution business, but the design of ViaSpan(R) is not based on modern molecular biology investigations. BLT has launched an aggressive program to determine the molecular basis of cell death during extended hypothermic and cryopreservation storage so that knowledge of these events can lead to a new generation of hypothermic solutions. As a result of BLT's early success, HTS is currently being used in a pancreatic islet transplantation Phase I clinical trial. DNA gels presented in this proposal demonstrate that cells cold stored for too long or cryopreserved die by apoptosis (programmed cell death) two days after they are returned to normothermic (37 degrees C) temperatures. HTS supplemented with apoptosis inhibitors blocks this process and improves HTS's performance. BLT currently has a patent pending that will allow BLT the exclusive right to formulate future HTS solutions by design so that apoptosis is inhibited. The Specific Aims of this Phase I project are to (a) determine the relative contributions of apoptosis and necrosis as a consequence of preservation-induced cellular injury to blood vessel cells; (b) determine which apoptosis inhibitors are the most effective at increasing HTS efficacy; (c) determine the relationship, if any, between necrosis/apoptosis, cytochrome C leakage from mitochondria, and loss of mitochondrial transmembrane potential; and (d) determine if extended preservation shortens telomeres and as a consequence precipitates premature cell senescence. Phase II studies will be devoted to HTS preservation of intact human blood vessels - work to be accomplished with our collaborator, CryoLife Inc. PROPOSED COMMERCIAL APPLICATIONS: BioLife is developing a new generation of preservation solutions that will improve the long term storage of human blood vessels used for clinical cardiovascular applications. The solutions will also facilitate preservation of future, tissue engineered blood vessels currently under development.